The goal of this project is to determine the agonist induced cellular mechanisms responsible for beta2-adrenergic receptor internalization. Beta2-adrenergic receptors apparently internalize during at least two different independent processes, down regulation and sequestration, which may be common to all G-protein coupled receptors. Sequestration has been implicated in the processes of short term receptor desensitization and resensitization. Loss of normal receptor function from failure of any component part of the sequestration pathway may contribute to disease as in chronic heart failure where normal cardiac contractility may depend upon normal receptor resensitization, or prevention of abnormal receptor desensitization. Various mutant beta2-adrenergic receptors have been produced which exhibit sequestration behavior intermediate between wild type and nonfunctional receptors by making point mutations in a motif at the junction of the seventh transmembrane, cytoplasmic region of the receptor. This motif is common to most of the known G-protein coupled receptors with only a few exceptions. The mechanisms governing beta- adrenergic receptor internalization then may be common to or serve as a paradigm to explain the agonist induced behavior of other members of the G-coupled protein receptor family. This study will test the ability of wild type and sequestration abnormal receptors to interact with other components of the cell, including betaARK1, a protein kinase that may play a role in chronic heart disease. Additionally, the generality of this paradigm will be assessed by comparison to other G-protein coupled receptors. Modified receptors that have been epitope tagged at their amino termini will be expressed in a transient or permanent manner in different cell lines using plasmid expression vectors. Receptor behavior and distribution will be studied either by radioligand binding with appropriate antagonists or monoclonal antibodies using either flow cytometry for quantitation or digital analysis of immunofluorescence cell images. Agonist dependent and independent receptor distribution will be correlated using antibodies to known receptor associated proteins such as beta-arrestin, betaARK and against other membrane proteins such as clathrin and caveolin in either whole cells using immunofluorescence or in purified cell lysates. Using the technique of co-immunoprecipitation with wild type and sequestration defective receptor, attempts will be made to determine other cell constituents that might be required for sequestration to occur normally. Identification of these components may facilitate the development of therapies aimed at reversing abnormal receptor behavior.